Splicing Programs and Cancer

Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets

The proto-oncogenic protein TAL1 controls TGF-β1 signaling through interaction with SMAD3

AbstractTGF-β1 is involved in many aspects of tissue development and homeostasis including hematopoiesis. The TAL1 transcription factor is also an important player of this latter process and is expressed very early in the myeloid and erythroid lineages. We previously established a link between TGF-β1 signaling and TAL1 by showing that the cytokine was able to induce its proteolytic degradation by the ubiquitin proteasome pathway. In this manuscript we show that TAL1 interacts with SMAD3 that acts in the pathway downstream of TGF-β1 association with its receptor. TAL1 expression strengthens the positive or negative effect of SMAD3 on various genes. Both transcription factors activate the inhibitory SMAD7 factor through the E box motif present in its transcriptional promoter. DNA precipitation assays showed that TAL1 present in Jurkat or K562 cells binds to this SMAD binding element in a SMAD3 dependent manner. SMAD3 and TAL1 also inhibit several genes including ID1, hTERT and TGF-β1 itself. In this latter case TAL1 and SMAD3 can impair the positive effect exerted by E47. Our results indicate that TAL1 expression can modulate TGF-β1 signaling by interacting with SMAD3 and by increasing its transcriptional properties. They also suggest the existence of a negative feedback loop between TAL1 expression and TGF-β1 signaling

A new advance in alternative splicing databases: from catalogue to detailed analysis of regulation of expression and function of human alternative splicing variants

<p>Abstract</p> <p>Background</p> <p>Most human genes produce several transcripts with different exon contents by using alternative promoters, alternative polyadenylation sites and alternative splice sites. Much effort has been devoted to describing known gene transcripts through the development of numerous databases. Nevertheless, owing to the diversity of the transcriptome, there is a need for interactive databases that provide information about the potential function of each splicing variant, as well as its expression pattern.</p> <p>Description</p> <p>After setting up a database in which human and mouse splicing variants were compiled, we developed tools (1) to predict the production of protein isoforms from these transcripts, taking account of the presence of open reading frames and mechanisms that could potentially eliminate transcripts and/or inhibit their translation, i.e. nonsense-mediated mRNA decay and microRNAs; (2) to support studies of the regulation of transcript expression at multiple levels, including transcription and splicing, particularly in terms of tissue specificity; and (3) to assist in experimental analysis of the expression of splicing variants. Importantly, analyses of all features from transcript metabolism to functional protein domains were integrated in a highly interactive, user-friendly web interface that allows the functional and regulatory features of gene transcripts to be assessed rapidly and accurately.</p> <p>Conclusion</p> <p>In addition to identifying the transcripts produced by human and mouse genes, fast DB <url>http://www.fast-db.com</url> provides tools for analyzing the putative functions of these transcripts and the regulation of their expression. Therefore, fast DB has achieved an advance in alternative splicing databases by providing resources for the functional interpretation of splicing variants for the human and mouse genomes. Because gene expression studies are increasingly employed in clinical analyses, our web interface has been designed to be as user-friendly as possible and to be readily searchable and intelligible at a glance by the whole biomedical community.</p

A novel androgen-regulated isoform of the TSC2 tumour suppressor gene increases cell proliferation

TSC2 (Tuberous sclerosis complex 2) is an important tumour suppressor gene, mutations within which are linked to the development of tuberous sclerosis and implicated in multiple tumour types. TSC2 protein complexes with TSC1 and blocks the ability of the Rheb (Ras homolog enriched in brain) GTPase to activate mTOR (mammalian target of rapamycin), a crucial signal transducer which regulates protein synthesis and cell growth. Here, we report the characterisation of a novel isoform of TSC2 which is under direct control of the ligand-activated androgen receptor. TSC2 isoform A (TSC2A) is derived from an internal androgen-regulated alternative promoter and encodes a 508-amino acid cytoplasmic protein corresponding to the C-terminal region of full-length TSC2, lacking the interaction domain for TSC1 and containing an incomplete interaction domain required for Rheb inactivation. Expression of TSC2A is induced in response to androgens and full-length TSC2 is co-ordinately down-regulated, indicating an androgen-driven switch in TSC2 protein isoforms. In contrast to the well-characterised suppressive effect on cell proliferation of full-length TSC2 protein, both LNCaP and HEK293 cells over-expressing TSC2 isoform A proliferate more rapidly (measured by MTT assays) and have increased levels of cells in S-phase (measured by both Edu staining and FACS analysis). Our work indicates, for the first time, a novel role for this well-known tumour suppressor gene, which encodes an activator of cell proliferation in response to androgen stimulation

Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

It is well established that transcription and alternative splicing events are functionally coupled during gene expression. Here, we report that protein arginine N-methyltransferase 6 (PRMT6) may play a key role in this coupling process by functioning as a transcriptional coactivator that can also regulate alternative splicing. PRMT6 coactivates the progesterone, glucocorticoid and oestrogen receptors in luciferase reporter assays in a hormone-dependent manner. In addition, small interfering RNA (siRNA) oligonucleotide duplex knockdown of PRMT6 disrupts oestrogen-stimulated transcription of endogenous GREB1 and progesterone receptor in MCF-7 breast cancer cells, demonstrating the importance of PRMT6 in hormone-dependent transcription. In contrast, the regulation of alternative splicing by PRMT6 is hormone independent. siRNA knockdown of PRMT6 increases the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor and spleen tyrosine kinase RNA transcripts in both the presence and absence of oestrogen. These results demonstrate that PRMT6 has a dual role in regulating gene expression and that these two functions can occur independently of each other

Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation [preprint]

mRNA translation and degradation are strongly interconnected processes that participate in the fine tuning of gene expression. Particularly, targeting mRNAs to translation-dependent degradation (TDD) could attenuate protein expression by making any increase in mRNA translation self-limiting. However, the extent to which TDD is a general mechanism for limiting protein expression is currently unknown. Here we describe a comprehensive analysis of basal and signal-induced TDD in mouse primary CD4 T cells. Our data indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner, both in resting and activated cells. Our analysis further identifies the length of untranslated regions, the density of ribosomes and the GC content of the coding region as major determinants of TDD magnitude. Consistent with this, all transcripts that undergo changes in ribosome density upon T cell activation display a corresponding change in their TDD level. Surprisingly, the amplitude of translation-independent mRNA decay (TID) appears as a mirror image of TDD. Moreover, TID also responds to changes in ribosome density upon T cell activation but in the opposite direction from the one observed for TDD. Our data demonstrate a strong interconnection between mRNA translation and decay in mammalian cells. Furthermore, they indicate that ribosome density is a major determinant of the pathway by which transcripts are degraded within cells

The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue

The regulation of ob gene expression in abdominal subcutaneous adipose tissue was investigated using a reverse transcription-competitive PCR method to quantify the mRNA level of leptin. Leptin mRNA level was highly correlated with the body mass index of 26 subjects (12 lean, 7 non-insulin-dependent diabetic, and 7 obese patients). The effect of fasting on ob gene expression was investigated in 10 subjects maintained on a hypocaloric diet (1045 KJ/d) for 5 d. While their metabolic parameters significantly changed (decrease in insulinemia, glycemia, and resting metabolic rate and increase in plasma ketone bodies), the caloric restriction did not modify the leptin mRNA level in the adipose tissue. To verify whether insulin regulates ob gene expression, six lean subjects underwent a 3-h euglycemic hyperinsulinemic (846 +/- 138 pmol/liter) clamp. Leptin and Glut 4 mRNA levels were quantified in adipose tissue biopsies taken before and at the end of the clamp. Insulin infusion produced a significant threefold increase in Glut 4 mRNA while leptin mRNA was not affected. It is concluded that ob gene expression is not acutely regulated by insulin or by metabolic factors related to fasting in human abdominal subcutaneous adipose tissue